Field of the Invention
The invention relates to a mass spectrometer with a laser desorption ion source, comprising a laser system for mass spectrometric analyses with ionization of the analyte molecules of a sample by matrix-assisted laser desorption.
Description of the Related Art
Over the past twenty years, two methods have gained acceptance in the mass spectrometry of biological macromolecules: matrix-assisted laser desorption and ionization (MALDI) and electrospray ionization (ESI). The biological macromolecules to be analyzed are termed analyte molecules below. In the MALDI method, the analyte molecules are generally prepared on the surface of a sample support in a solid, polycrystalline matrix layer, and are predominantly ionized with a single charge, whereas in the ESI method they are dissolved in a liquid and ionized with multiple charges. It was these two methods which made possible the mass spectrometric analysis of biological macromolecules in genomics, proteomics and metabolomics; their inventors, John B. Fenn and Koichi Tanaka, were awarded the Nobel Prize in Chemistry in 2002.
Matrix-assisted laser desorption and ionization (MALDI) has been improved enormously in recent years by switching from nitrogen lasers to solid state UV lasers with a longer service life, and in particular by using beam generation with a spatially modulated beam profile for an increased ion yield. The method of beam generation and the corresponding laser systems have been described in the equivalent documents DE 10 2004 044 196 A1, GB 2 421 352 B and U.S. Pat. No. 7,235,781 B2 (A. Haase et al., 2004) and have become known under the name “smart beam”. These documents are incorporated herein by reference.
The invention in the above-listed documents is based on the finding that the ion yield from a sample volume increases greatly if the laser spots are made very small, down to around five micrometers in diameter. This means, however, that energy densities very soon reach levels at which spontaneous fragmentation of the ionized molecules occurs. On the other hand, if one remains below this limit, too few ions are generated per shot from this small sample volume. As a solution, a pattern of several spots is proposed in order to obtain sufficient ions without fragmentation. It turns out that other parameters, such as the mass resolution, are also positively affected. With the fine spot pattern, hardly any sample material is spattered, something that is always a problem for larger spot diameters with larger amounts of molten material. Preferably around five to fifteen sharply focused laser spots with a diameter of around five micrometers should be produced to generate the right number of ions in each laser shot. Each laser spot should have the same energy density, since the ion generation rate increases at roughly the sixth to seventh power of the energy density in the laser spot. If the energy density for a spot were to be increased by 50 percent, for example, the degree of ionization would increase by more than a factor of ten. The other spots of the pattern would then produce hardly any analyte ions in comparison, but would consume sample in an undesirable way.
The generation of patterns increases the ion yield per analyte molecule by far more than a factor of 10 and reduces the sample consumption accordingly; this is important especially for imaging mass spectrometry on thin tissue sections. Since modern mass spectrometers are designed for spectrum acquisition rates of 10,000 image spectra per second and more, the generation of the spot pattern must additionally be very energy-efficient in order to obviate the need for expensive very high-performance lasers.
Generating a pattern with a few UV spots of the same energy density is not a trivial undertaking. A region with intensity peaks of equal intensity can be created with an arrangement of two matched lens arrays (“fly's eye”), (see, for example, “Refractive Micro-optics for Multi-spot and Multi-line Generation”, M. Zimmermann et al., Proceedings of the 9th International Symposium on Laser Precision Microfabrication; LPM2008). In the infrared, at a wavelength of 10 micrometers, this region can comprise precisely nine spots, but in the ultraviolet, it comprises hundreds of spots. Another possibility is to use diffractive beam splitters, but their production costs are high. Since fused silica has to be used for the optical elements at these wavelengths, it is usually very expensive to manufacture appropriate beam-shaping optical devices.
A method for the energy-efficient generation of only a few UV spots of equal energy density and the associated equipment are disclosed in the equivalent documents DE 10 2011 116 405 A1, U.S. Pat. No. 8,431,890 B2 and GB 2 495 815 A (A. Haase and J. Höhndorf). These documents are also incorporated herein by reference. These documents also contain a longer introduction to the current knowledge on MALDI and describe in detail the reason for the introduction of spot patterns.
The components for equipment in accordance with these documents are relatively expensive, however, and the components used must be adjusted very precisely and reproducibly. There is still a need for low-cost methods and equipment, and particularly ones that have not to be critically adjusted. The insensitivity to adjustment becomes particularly important when several pattern generators are to be used in rapid interchange in order to match the spot patterns to the analytical task.